A HTS Assay for Identifying Anti-Viral Drugs Against Arbovirus Infections

描述（由申请人提供）：在本提案中，我们建议转移我们的HTS就绪、基于荧光的半胱天冬酶-3/7测定，以基于BTV感染模型系统中不同的宿主凋亡反应来鉴定抗虫媒病毒感染的新型抗病毒药物。节肢动物传播病毒（虫媒病毒）是重要的人类和/或动物病原体，其引起急性病毒感染，伴随严重疾病和/或死亡。最近的几种人类和/或动物流行病是由虫媒病毒引起的，包括亚洲的登革病毒（DNV）、北美的西尼罗河病毒（WNV）和欧洲的蓝舌病毒（BTV）。虫媒病毒是唯一的，因为它们通过节肢动物载体传播给它们的脊椎动物宿主，因此，它们必须能够在两个非常不同的宿主类群-脊椎动物和昆虫中复制。虽然虫媒病毒在脊椎动物和昆虫细胞中都能有效复制，但各自的宿主细胞反应却大不相同。例如，BTV感染在脊椎动物细胞中诱导快速的凋亡反应，而这种凋亡反应在昆虫细胞中不明显，尽管在两种宿主细胞中都有生产性病毒复制。虽然细胞凋亡与受感染动物/人类的虫媒病毒疾病呈正相关，但昆虫没有显示出任何疾病的可检测迹象。很少有人注意到这种不同的宿主细胞反应的意义和适应这种策略的药物发现的脊椎动物宿主的保护的可能性。我们推测，化合物防止BTV诱导的脊椎动物细胞凋亡可以通过抑制细胞凋亡或通过干扰病毒的生命周期。第二次检测将使我们能够将抗病毒药物与凋亡抑制剂分开。这些抗病毒化合物可能也可以保护宿主细胞免受其他虫媒病毒（包括DNV和WNV）诱导的宿主凋亡反应。我们的长期目标是开发新的预防和控制人类虫媒病毒病的措施。本申请的目的将通过进行以下具体目标来实现：在脊椎动物细胞中的BTV感染模型中使用开发的有效HTS测定来筛选指定的化合物文库。基于我们的初步数据显示BTV通过内在凋亡途径诱导脊椎动物细胞凋亡，使用该模型系统设计、开发、优化和验证了有效的HTS测定。我们建议将该试验转移至指定的筛选中心，以重现、重复和自动化该试验。还将实施使用细胞凋亡诱导剂（包括星形孢菌素）的二次试验，以确认命中并排除假阳性（包括细胞凋亡抑制剂）。这些命中的可能机制也将被检查，以优先考虑这些命中进一步调查对DNV和WNV感染。节肢动物传播病毒（arboviruses）是重要的人类和/或动物病原体，其引起急性病毒感染，伴随严重疾病和/或死亡，几种虫媒病毒引起最近的人类或动物流行病，包括亚洲的登革热病毒、北美的西尼罗河病毒和欧洲的蓝舌病毒。在该提案中，我们提出转移我们的HTS就绪的、基于荧光的半胱天冬酶-3/-7测定，以基于虫媒病毒感染中的不同宿主凋亡反应来鉴定针对虫媒病毒感染的新靶标，以保护宿主免受虫媒病毒感染，包括登革病毒和蓝舌病毒。我们的长期目标是开发新的预防和控制人类和动物虫媒病毒病的措施。

DESCRIPTION (provided by applicant): In this proposal, we propose to transfer our HTS-ready, florescence based caspases-3/7 assay to identify novel anti-viral drugs against arboviruses infection based on the different host apoptotic responses in BTV infection model system. Arthropod borne viruses (arboviruses) are important human and/or animal pathogens that cause acute virus infections with severe diseases and/or death. Several recent human and/or animal epidemics are caused by arboviruses, including Dengue virus (DNV) in Asia, West Nile virus (WNV) in North America and Bluetongue virus (BTV) in Europe. Arboviruses are unique because they are transmitted to their vertebrate hosts by arthropod vectors, therefore, they must be capable of replicating in two very divergent host taxa--vertebrates and insects. Although arboviruses replicate efficiently in both vertebrate and insect cells, the respective host cellular responses are quite different. For example, BTV infection induces a rapid apoptotic response in vertebrate cells, whereas such apoptotic response is unapparent in insect cells, despite the productive virus replication in both host cells. While apoptosis has been positively linked to arbovirus diseases in infected animals/human, insects show no detectable signs of any diseases. Little attention has been paid to the significance of such different host cellular responses and the possibility of adapting such strategy for drug discovery to the protection of vertebrate hosts. We hypothesize that compounds preventing BTV-induced apoptosis in vertebrate cells could act either via inhibiting apoptosis or via interfering viral life-cycle. A secondary assay will allow us to separate anti-viral hits from the apoptosis inhibitors. These anti-viral compounds could presumably also protect host cells from host apoptotic response induced by other arboviruses, including DNV and WNV. Our long term goal is to develop new prevention and control measures for arbovirus diseases in human. The objectives of this application will be achieved by carrying out the following Specific Aim: To screening designated compound library using the developed efficient HTS assay in the BTV infection model in vertebrate cells. Based on our preliminary data showing that BTV-induced apoptosis in vertebrate cells via intrinsic apoptotic pathway, an efficient HTS assay have been designed, developed, optimized and validated using this model system. We propose to transfer this assay to the designated screening center to reproduce, miniaturize and automate the assay. A secondary assay using apoptosis inducers including Staurosporine will also be implemented to confirm hits and exclude false positives including apoptosis inhibitors. The possible mechanism of these hits will also be examined to prioritize these hits for further investigations against DNV and WNV infection. Arthropod borne viruses (arboviruses) are important human and/or animal pathogens that cause acute virus infections with severe diseases and/or death, several arboviruses cause recent human or animal epidemics, including Dengue virus in Asia, West Nile virus in North America and Bluetongue virus in Europe. In this proposal, we proposed to transfer our HTS-ready, florescence based caspases- 3/-7 assay to identify novel targets against arboviruses infection based on the different host apoptotic responses in arbovirus infection to protect hosts from arboviruses infections, including Dengue virus and Bluetongue virus. Our long term goal is to develop new prevention and control measures for arbovirus diseases in human and animals.